Cyanine 3.5 (Cy3.5) is a fluorescent dye that belongs to the Cyanine family of synthetic polymethine dyes. Cy3.5 is reactive, water-soluble, and has an absorbance maximum of 581 nm and an emission maximum of 596 nm. It is available as a phosphoramidite, and is used to fluorescently label oligonucleotides at either the 5’- or 3’-end, or internally. Cy3.5 plays a particularly important role in real-time PCR applications, being used as a reporter moiety in TaqMan probes (1), Scorpion primers (2) and Molecular Beacons (3). For such probes, Cy3.5 is most commonly paired with the dark quencher BHQ-2, as the two have excellent spectral overlap.
Cy3 can also be used to label DNA oligos for use as hybridization probes in other applications, such as Fluorescent In-Situ Hybridization (FISH).
1. Livak, K.J., Flood, S.J.A., Marmaro, J., Giusti, W., Deetz, K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization.PCR Methods Appl. (1995), 4: 1-6.
2. Thelwell, N., Millington, S., Solinas, A., Booth, J., Brown, T. Mode of action and application of Scorpion primers to mutation detection. Nucleic Acids Res. (2000), 28: 3752-3761.
3. Tyagi, S., Kramer, F.R. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. (1996), 14: 303-308.